The long-term goal of this research program is to develop antibody therapies that eradicate tumors by specifically activating selected cellular components of the innate immune system, with an emphasis on natural killer (NK) cells. Our work has focused on the creation and development of antibodies that target tumor antigens and the cytotoxicity trigger molecule, CD16, the low affinity Fcy receptor for aggregated immunoglobulin. Improved understanding of the biology of natural killer cells has led to the identification of many of the receptors involved in the decision of NK cells to lyse or ignore a given cellular target. Moreover, many of the signaling elements required to implement these decisions have been elucidated. The underlying hypothesis to be tested in this proposal is that antibodies can be employed to selectively activate NK cell cytotoxicity at tumor sites. The proposed experiments will identify candidate molecules and strategies for manipulation of NK cell cytotoxicity with a goal of clinically developing the most promising strategies. The first specific aim is to promote tumor lysis by human natural killer cells through the activation of the Fc7 receptor, CD16. We will test the hypothesis that multivalent, high-avidity interactions with tumor cells and monovalent, low affinity binding to a natural killer cell activating receptor by a human antibody will yield efficient in vivo anti-tumor effects. A trimeric, bispecific minibody (TriBi) containing two moderate-affinity (10 -9 M) binding sites for HER2/neu and a single, low-affinity (10 -7 M) binding site for CD16 will be evaluated for its in vivo properties to determine the value of increased size and multivalent binding on tumor targeting and anti-tumor effects in three distinct murine models. The second specific aim is to enhance tumor-targeted human natural killer cell activation using antibodies that bind to HER2/neu and to the NKG2D NK cell activating receptor. We hypothesize that NK activation can be enhanced by activating receptors that signal through multiple membrane- proximal pathways. NKG2D is an attractive candidate activation receptor because it is broadly expressed by NK cells, and signals through DAP10, whereas CD16 signals through FceRy and CD3_ chains. Multifunctional antibodies that bind multivalently to HER2/neu and with low affinity to either CD16 or NKG2D (or to both molecules) should be efficient and effective approaches for the tumor-selective activation of NK cells. Since NK cells may efficiently traffic into tumors, the third specific aim is to target NK cells to tumor-associated endothelial cell molecules (TEM), damaging tumor blood vessel integrity by triggering NK cell activation through CD16 or NKG2D. TEM represent potentially important targets for diverse therapeutic strategies. We propose to test the hypothesis that the selective, tumor-restricted localization and activation of NK cells based on targeting tumor-related endothelium will improve NK cell accumulation in tumor-related blood vessels and at tumor cells, and will amplify the therapeutic benefits of NK cell-related cytotoxicity. The successful execution of these studies will identify candidate strategies for the clinical exploitation of tumor-selective NK cell activation.